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Biochemical Pharmacology (v.81, #3)

Editorial Advisory Board (pp. iii).

Anti-angiogenic effects of dietary isothiocyanates: Mechanisms of action and implications for human health by Breeze E. Cavell; Sharifah S. Syed Alwi; Alison Donlevy; Graham Packham (pp. 327-336).

Isothiocyanates (ITCs) are electrophilic compounds derived from plants and are thought to play a major role in the potential chemopreventive effects associated with high intake of cruciferous vegetables. ITCs are also being evaluated for chemotherapeutic activity in early phase clinical trials. In addition to their effects on carcinogen metabolism and cancer cell survival and proliferation, ITCs have been shown to effectively interfere with angiogenesis in vitro and in vivo. Angiogenesis is the development of a new blood supply from existing vasculature and is required for tumours to develop beyond a small size limit determined by the diffusion limit for oxygen. Inhibition of angiogenesis may play a key role in the potential chemopreventive/chemotherapeutic activity of ITCs. In this review we highlight recent data demonstrating that ITCs have anti-angiogenic activity and identify potential molecular targets for these effects, including hypoxia-inducible factor (HIF), nuclear factor κB (NF-κB), activator protein 1 (AP1) and tubulin. We also discuss these findings in light of the potential chemopreventive/chemotherapeutic effects of ITCs.

Keywords: Abbreviations; 4E-BP1; translation factor 4E binding protein 1; AITC; allyl isothiocyanate; Ang2; angiopoietin 2; AP1; activator protein 1; bFGF; basic fibroblast-like growth factor; BITC; benzyl isothiocyanate; BNIP3; BCL2/adenovirus E1B 19; kDa interacting protein; CAIX; carbonic anhydrase IX; CBP; CREB binding protein; eIF4A; eukaryotic translation initiation factor 4A; eIF4E; eukaryotic translation initiation factor 4E; eIF4F; eukaryotic translation initiation factor 4F; eIF4G; eukaryotic translation initiation factor 4G; FIH; factor inhibiting HIF1; GLUT1; glucose transporter type 1; GSH; glutathione; GST; glutathione-; S; -transferases; HDAC; histone deacetylases; HIF; hypoxia inducible factor; HRE; hypoxia-response elements; HUVEC; human umbilical vein endothelial cells; IL8; interleukin 8; ITC; isothiocyanate; JNK; Jun N-terminal kinase; Keap1; kelch-like ECH-associated protein 1; mTOR; mammalian target of rapamycin; NAC; N; -acetylcysteine; NF-κB; nuclear factor κB; PHD; prolyl hydoxylases; PEITC; phenethyl isothiocyanate; PGK1; phosphoglycerate kinase 1; ROS; reactive oxygen species; SFN; sulforaphane; TGFβ; transforming growth factor β; VEGF; vascular endothelial growth factor; UTR; untranslated region; VHL; von Hippel–Landau proteinKey words; Isothiocyanates; Angiogenesis; Hypoxia inducible factor; Nuclear factor κB; Tubulin

Pharmacological and dietary modulators of paraoxonase 1 (PON1) activity and expression: The hunt goes on by Lucio G. Costa; Gennaro Giordano; Clement E. Furlong (pp. 337-344).

Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated enzyme displaying esterase and lactonase activity. PON1 hydrolyzes several organophosphorus (OP) insecticides and nerve agents, a number of exogenous and endogenous lactones, and metabolizes toxic oxidized lipids of low density lipoproteins (LDL) and HDL. As such, PON1 plays a relevant role in determining susceptibility to OP toxicity, cardiovascular diseases and several other diseases. Serum PON1 activity in a given population can vary by at least 40-fold. Most of this variation can be accounted for by genetic polymorphisms in the coding region (Q192R, L55M) and in the promoter region (T-108C). However, exogenous factors may also modulate PON1 activity and/or level of expression. This paper examines various factors that have been found to positively modulate PON1. Certain drugs (e.g. hypolipemic and anti-diabetic compounds), dietary factors (antioxidants, polyphenols), and life-style factors (moderate alcohol consumption) appear to increase PON1 activity. Given the relevance of PON1 in protecting from certain environmental exposure and from cardiovascular and other diseases, there is a need for further mechanistic, animal, and clinical research in this area, and for consideration of possible alternative strategies for increasing the levels and activity of PON1.

Keywords: Key words; Paraoxonase 1; Statins; Hypolipemic drugs; Anti-diabetic drugs; Antioxidants; Polyphenols; Alcohol

Sodium salicylate is a novel catalytic inhibitor of human DNA topoisomerase II alpha by Jason T. Bau; Ebba U. Kurz (pp. 345-354).

SS pretreatment prevents doxorubicin-induced DNA double-stranded breaks and prevents stabilization of cleavable complexes • Does not involve NFκB or COX-2 • SS pretreatment reduces cellular sensitivity to topoisomerase II poisons.We have previously reported that pretreatment of human lymphoblastoid cells with the hydroxyl radical scavenger, N-acetyl cysteine, attenuates doxorubicin-induced DNA damage signalling through the ATM protein kinase. We sought to extend these studies to examine the effects of other hydroxyl radical scavengers in human breast cancer cells. Using MCF-7 cells, we observed that doxorubicin treatment triggered autophosphorylation of ATM on serine 1981 and the ATM-dependent activation of its downstream effectors p53, Chk2, and SMC1. Furthermore, we demonstrate that this effect was attenuated by pretreatment of cells with the hydroxyl radical scavengers sodium benzoate, sodium salicylate and, to a lesser extent, N-acetyl cysteine, but not Trolox™. Intriguingly, these effects were independent of doxorubicin's ability to redox cycle, were observed with multiple classes of topoisomerase II poisons, but did not represent a general damage-attenuating response. In addition, the observed effects were independent of the ability of sodium salicylate to inhibit cyclooxygenase-2 or NFκB. We demonstrate that sodium salicylate prevented doxorubicin-induced DNA double-strand break generation, which was attributable to inhibition of doxorubicin-stabilized topoisomerase IIα-DNA cleavable complex formation in vivo. Using topoisomerase IIα-DNA cleavage and decatenation assays, we determined that sodium salicylate is a catalytic inhibitor of topoisomerase IIα. Consistent with the observed inhibition of double-strand break formation, pretreatment of cells with sodium salicylate attenuated doxorubicin and etoposide cytotoxicity. These results demonstrate a novel mechanism of action for sodium salicylate and suggest that further study on the mechanism of topoisomerase II inhibition and the effects of related therapeutics on doxorubicin and etoposide cytotoxicity are warranted.

Keywords: Abbreviations; 5-IDNR; 5-iminodaunorubicin; ASA; acetylsalicylic acid; ATM; ataxia telangiectasia mutated; COX-2; cyclooxygenase-2; CPT; camptothecin; DEM; diethyl maleate; DSB; double-stranded break; ICE; in vivo complex of enzyme; IR; ionizing radiation; kDNA; kinetoplast DNA ;; MTX; mitoxantrone; NAC; N-acetyl cysteine; NFκB; nuclear factor kappa B; NSAID; non-steroidal anti-inflammatory drug; PIKKs; phosphoinositide 3-kinase-like kinases; SS; sodium salicylate; TNFα; tumor necrosis factor alpha; topo II; topoisomerase IIα; TPT; topotecan; VP-16; etoposideTopoisomerase IIα; Sodium salicylate; Catalytic inhibitor; DNA damage signalling; Doxorubicin

Inhibitors of NQO1: Identification of compounds more potent than dicoumarol without associated off-target effects by Katherine Ann Scott; John Barnes; Roger Clive Whitehead; Ian James Stratford; Karen Ann Nolan (pp. 355-363).

Differential effects of dicoumarol analogues as inhibitors of NQO1, production of superoxide and induction of apoptosis and toxicity in cancer cells.The enzyme NAD(P)H quinone oxidoreductase (NQO1) can function both as a detoxifying enzyme as well as chaperone protein. The latter property has been extensively characterized by the use of dicoumarol which inhibits the chaperone properties of NQO1 in cells. However, the use of this compound is compromised by its multiple “off-target” effects. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of NQO1 in cells have been identified (Nolan et al., Biochem Pharmacol 2010;80:977–81). The purpose of the work reported here is to evaluate the off-target effects of these compounds when compared to dicoumarol. A range of these substituted coumarins are identified that are significantly less toxic than dicoumarol in a panel of nine cell lines. Further a number of the compounds generate much less intracellular superoxide, and many of them also show a reduced ability to induce apoptosis when compared to dicoumarol. None of these effects correlate with the ability of the compounds to inhibit the enzymatic activity of NQO1 in cells. In conclusion, potent inhibitors of NQO1 have been identified that will be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.

Keywords: NQO1; Dicoumarol; Coumarins; Superoxide; Apoptosis

Sustained exposure to the DNA demethylating agent, 2′-deoxy-5-azacytidine, leads to apoptotic cell death in chronic myeloid leukemia by promoting differentiation, senescence, and autophagy by Michael Schnekenburger; Cindy Grandjenette; Jenny Ghelfi; Tommy Karius; Bernard Foliguet; Mario Dicato; Marc Diederich (pp. 364-378).

In addition to its demethylating properties, 2′-deoxy-5-azacytidine (DAC) induces cell cycle arrest, differentiation, cell sensitization to chemotherapy, and cell death. However, the mechanisms by which DAC induces antiproliferation via these processes and how they are interconnected remain unclear. In this study, we found that a clinically relevant concentration of DAC triggered erythroid and megakaryocytic differentiation in the human chronic myeloid leukemia (CML) K-562 and MEG-01 cell lines, respectively. In addition, cells showed a marked increase in cell size in both cell lines and a more adhesive cell profile for MEG-01. Furthermore, DAC treatment induced cellular senescence and autophagy as shown by β-galactosidase staining and by autophagosome formation, respectively. After prolonged DAC treatment, phosphatidyl serine exposure, nuclear morphology analysis, and caspase cleavage revealed an activation of mitochondrial-dependent apoptosis in CML cells. This activation was accompanied by a decrease of anti-apoptotic proteins and an increase of calpain activity. Finally, we showed that combinatory treatment of relatively resistant CML with DAC and either conventional apoptotic inducers or with an histone deacetylase inhibitor increased synergistically apoptosis. We therefore conclude that induction of differentiation, senescence, and autophagy in CML are a key in cell sensitization and DAC-induced apoptosis.

Keywords: 5-Aza-2′-deoxycytidine; Chronic myeloid leukemia; Differentiation; Senescence; Autophagy; Apoptosis

Prostaglandin E₂ regulates cellular migration via induction of vascular endothelial growth factor receptor-1 in HCA-7 human colon cancer cells by Hiromichi Fujino; Kaori Toyomura; Xiao-bo Chen; John W. Regan; Toshihiko Murayama (pp. 379-387).

An important event in the development of tumors is angiogenesis, or the formation of new blood vessels. Angiogenesis is also known to be involved in tumor cell metastasis and is dependent upon the activity of the vascular endothelial growth factor (VEGF) signaling pathway. Studies of mice in which the EP3 prostanoid receptors have been genetically deleted have shown a role for these receptors in cancer growth and angiogenesis. In the present study, human colon cancer HCA-7 cells were used as a model system to understand the potential role of EP3 receptors in tumor cell migration. We now show that stimulation of HCA-7 cells with PGE₂ enhanced the up-regulation of VEGF receptor-1 (VEGFR-1) expression by a mechanism involving EP3 receptor-mediated activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Moreover, the PGE₂ stimulated increase in VEGFR-1 expression was accompanied by an increase in the cellular migration of HCA-7 cells. Given the known involvement of VEGFR-1 in cellular migration, our results suggest that EP3 receptors may contribute to tumor cell metastasis by increasing cellular migration through the up-regulation of VEGFR-1 signaling.

Keywords: Abbreviations; PGE; ₂; prostaglandin E; ₂; VEGF; vascular endothelial growth factor; VEGFR; VEGF receptor; DMEM; Dulbecco's modified Eagle's medium; RT; reverse transcription; PCR; polymerase chain reaction; GAPDH; glyceraldehydes-3-phosphate dehydrogenase; ERKs; extracellular signal-regulated kinases; MEK; mitogen-activated protein kinase/ERK kinase; PTX; pertussis toxin; BSA; bovine serum albumin; PI3 kinase; phosphatidylinositol 3-kinase; FGFR-1; fibroblast growth factor receptor-1; EGFR; epidermal growth factor receptorPGE; ₂; Human EP3 receptor; VEGFR-1; Colon cancer; Metastasis

Recombinant chimeric lectins consisting of mannose-binding lectin and L-ficolin are potent inhibitors of influenza A virus compared with mannose-binding lectin by Wei-Chuan Chang; Kevan L. Hartshorn; Mitchell R. White; Patience Moyo; Ian C. Michelow; Henry Koziel; Bernard T. Kinane; Emmett V. Schmidt; Teizo Fujita; Kazue Takahashi (pp. 388-395).

MBL structurally contains a type II-like collagenous domain and a carbohydrate recognition domain (CRD). We have recently generated three novel recombinant chimeric lectins (RCL), in which varying length of collagenous domain of mannose-binding lectin (MBL) is replaced with that of L-ficolin (L-FCN). CRD of MBL is used for target recognition because it has a broad spectrum in pathogen recognition compared with L-FCN. Results of our study demonstrate that these RCLs are potent inhibitors of influenza A virus (IAV). RCLs, against IAV, show dose-dependent activation of the lectin complement pathway, which is significantly higher than that of recombinant human MBL (rMBL). This activity is observed even without MBL-associated serine proteases (MASPs, provided by MBL deficient mouse sera), which have been thought to mediate complement activation. These observations suggest that RCLs are more efficient in associating with MASP-2, which predominantly mediates the activity. Yet, additional serum further increases the activity while RCL-mediated coagulation-like enzyme activities are diminished compared with rMBL, suggesting reduced association with MASP-1, which has been shown to mediate coagulation-like activity. These data suggest that RCLs may interfere less with host coagulation, which is advantageous to be a therapeutic drug. Importantly, these RCLs have surpassed rMBL for anti-viral activities, such as viral aggregation, reduction of viral hemagglutination (HA) and inhibition of virus-mediated HA and neuraminidase (NA) activities. These results are encouraging that novel RCLs could be used as anti-IAV agents with less side effect and that RCLs would be suitable candidates in developing a new anti-IAV therapy.

Keywords: Innate immunity; Mannose-binding lectin; Ficolin; Influenza A virus; Complement; Coagulation

Cross-talk between minimally primed HL-60 cells and resting HUVEC reveals a crucial role for adhesion over extracellularly released oxidants by M. Allegra; F. D’Acquisto; L. Tesoriere; M.A. Livrea; M. Perretti (pp. 396-401).

This study demonstrates that a long-lasting co-culture of neutrophil surrogates (HL-60 cells), minimally primed by platelet activating factor (PAF), and resting endothelial cells (EC) results in the elaboration of an hyper-adhesive endothelial surface, as measured by the increase in the expression of endothelial adhesion molecules E-Selectin, VCAM-1, and ICAM-1. This endothelial dysfunction is mediated by the activation of the redox-sensitive transcription factor NF-κB through an exclusive adhesion-driven mechanism active in the endothelial cell: reactive oxygen and nitrogen species, extracellularly released by minimally primed HL-60 cells, are not involved in the induction of the endothelial dysfunction.By exploring for the first time the potential for minimally primed neutrophil surrogates to induce endothelial dysfunction, this study suggests a novel mechanism which may be operative in pathologies, mediated by minimally primed neutrophils, such as hyperdyslipidemia and cardiovascular complications.

Keywords: Neutrophils; Priming; Endothelial cells; Inflammation; Adhesion; Oxidants

Stable formyl peptide receptor agonists that activate the neutrophil NADPH-oxidase identified through screening of a compound library by Huamei Forsman; Christina Kalderén; Anna Nordin; Erik Nordling; Annika Jernmalm Jensen; Claes Dahlgren (pp. 402-411).

The neutrophil formyl peptide receptors (FPR1 and FPR2) are G-protein coupled receptors that can induce pro-inflammatory as well as anti-inflammatory activities when activated. Accordingly, these receptors may become therapeutic targets for the development of novel drugs to be used for reducing the inflammation induced injuries in asthma, rheumatoid arthritis, Alzheimer's disease, cardiovascular diseases and traumatic shock. We screened a library of more then 50K small compounds for an ability of the compounds to induce a transient rise in intracellular Ca²⁺ in cells transfected to express FPR2 (earlier called FPRL1 or the lipoxin A₄ receptor). Ten agonist hits were selected for further analysis representing different chemical series and five new together with five earlier described molecules were further profiled. Compounds 1–10 gave rise to a calcium response in the FPR2 transfectants with EC₅₀ values ranging from 4×10⁻⁹M to 2×10⁻⁷M. All 10 compounds activated human neutrophils to release superoxide, and based on the potency of their activity, the three most potent activators of the neutrophil NADPH-oxidase were further characterized. These three agonists were largely resistant to inactivation by neutrophil produced reactive oxygen species and shown to trigger the same functional repertoire in neutrophils as earlier described peptide agonists. Accordingly they induced chemotaxis, granule mobilization and secretion of superoxide. Interestingly, the oxidase activity was largely inhibited by cyclosporine H, an FPR1 selective antagonist, but not by PBP10, an FPR2 selective inhibitor, suggesting that FPR1 is the preferred receptor in neutrophils for all three agonists.

Keywords: Neutrophils; Formyl peptide receptors; Receptor antagonists; Signal transduction; NADPH-oxidase; Chemoattractant receptors

NFκB inhibitors induce cell death in glioblastomas by Alfeu Zanotto-Filho; Elizandra Braganhol; Rafael Schröder; Luís Henrique T. de Souza; Rodrigo J.S. Dalmolin; Matheus A. Bittencourt Pasquali; Daniel Pens Gelain; Ana Maria Oliveira Battastini; José Cláudio Fonseca Moreira (pp. 412-424).

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy.

Keywords: NFκB; Glioblastoma; NFκB inhibitors; Apoptosis; Chemotherapy

DrTx(1–42), a C-terminally truncated analogue of drosotoxin, is a candidate of analgesic drugs by Shunyi Zhu; Lan Luo; Ping Li; Bin Gao; Limei Zhu; Yuzhe Yuan (pp. 425-431).

The C-terminally truncated analogue of drosotoxin (DrTx(1–42)) retains the blocking activity of the intact molecule on TTX-R sodium channels.Drosotoxin is an engineered tetrodotoxin-resistant (TTX-R) sodium channel-specific blocker with a non-toxic structural core (Zhu et al. Biochem Pharmacol 2010; 80:1296–302). Here, we report the discovery and functional characterization of a carboxyl-terminally truncated analogue of drosotoxin (named DrTx(1–42)) which selectively inhibited dorsal root ganglion (DRG) neuron TTX-R sodium current ( I_Na) with an IC₅₀ value of 1.74±0.07μM. Consistent with this effect, DrTx(1–42) significantly attenuates inflammatory hyperalgesia of mice in a formalin-induced pain model with stronger potency than indomethacin, a nonsteroidal anti-inflammatory and analgesic drug. Our mutational experiments indicate that the N-turn insertion is an essential functional determinant for the emergence of neurotoxicity from a non-toxic antifungal scaffold.

Keywords: Sodium channel; Tetrodotoxin; Inflammatory hyperalgesia; Drosotoxin

The role of CYP3A4 in amiodarone-associated toxicity on HepG2 cells by Anja Zahno; Karin Brecht; Réjane Morand; Swarna Maseneni; Michael Török; Peter W. Lindinger; Stephan Krähenbühl (pp. 432-441).

Amiodarone is a class III antiarrhythmic drug with potentially life-threatening hepatotoxicity. Recent in vitro investigations suggested that the mono-N-desethyl (MDEA) and di-N-desethyl (DDEA) metabolites may cause amiodarone's hepatotoxicity. Since cytochrome P450 (CYP) 3A4 is responsible for amiodarone N-deethylation, CYP3A4 induction may represent a risk factor. Our aim was therefore to investigate the role of CYP3A4 in amiodarone-associated hepatotoxicity. First, we showed that 50μM amiodarone is more toxic to primary human hepatocytes after CYP induction with rifampicin. Second, we overexpressed human CYP3A4 in HepG2 cells (HepG2 cells/CYP3A4) for studying the interaction between CYP3A4 and amiodarone in more detail. We also used HepG2 wild type cells (HepG2 cells/wt) co-incubated with human CYP3A4 supersomes for amiodarone activation (HepG2 cells/CYP3A4 supersomes). Amiodarone (10–50μM) was cytotoxic for HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes, but not for HepG2 cells/wt or less toxic for HepG2 cells/wt incubated with control supersomes without CYP3A4. Co-incubation with ketoconazole, attenuated cytotoxicity of amiodarone incubated with HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes. MDEA and DDEA were formed only in incubations containing HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes but not by HepG2 cells/wt or HepG2 cells/wt with control supersomes. Metabolized amiodarone triggered the production of reactive oxygen species, induced mitochondrial damage and cytochrome c release, and promoted apoptosis/necrosis in HepG2 cells/CYP3A4, but not HepG2 cells/wt. This study supports the hypothesis that a high CYP3A4 activity is a risk factor for amiodarone's hepatotoxicity. Since CYP3A4 inducers are used frequently and amiodarone-associated hepatotoxicity can be fatal, our observations may be clinically relevant.

Keywords: Amiodarone; N-mono-desethylamiodarone; N-di-desethylamiodarone; HepG2 cells; CYP3A4

CYP1D1, pseudogenized in human, is expressed and encodes a functional drug-metabolizing enzyme in cynomolgus monkey by Yasuhiro Uno; Shotaro Uehara; Norie Murayama; Hiroshi Yamazaki (pp. 442-450).

Cytochrome P450 (P450 or CYP) 1 family consists of the CYP1A, CYP1B, CYP1C, and CYP1D subfamilies. In the human genome, CYP1A1, CYP1A2, and CYP1B1 are expressed and encode functional enzymes, whereas CYP1D1P (formerly known as CYP1A8P) is present as a pseudogene due to five nonsense mutations in the putative coding region. In this study, we identified CYP1D1 cDNA, highly identical (nearly 95%) to human CYP1D1P sequence, in cynomolgus monkey, a species frequently used in drug metabolism studies due to its evolutionary closeness to human. The amino acid sequence deduced from cynomolgus monkey CYP1D1 cDNA shared the high sequence identity (91%) with human CYP1D1P (postulated from the gene sequence), and the highest sequence identity (44–45%) with CYP1A1 and CYP1A2 among cynomolgus monkey P450s. CYP1D1 mRNA was most abundantly expressed in liver, followed by kidney, and jejunum. The hepatic expression level of CYP1D1 mRNA was comparable to that of CYP1A1 mRNA and much higher than that of CYP1A2 mRNA. CYP1D1 was barely detectable in immunoblots of cynomolgus monkey liver. Cynomolgus monkey CYP1D1 mRNA was induced in primary hepatocytes with omeprazole. Cynomolgus monkey CYP1D1 protein heterologously expressed in Escherichia coli catalyzed ethoxyresorufin O-deethylation and caffeine 8-hydroxylation, which CYP1As also catalyze. Finally, no nonsense mutations, corresponding to those found in human CYP1D1P, were found in the 20 cynomolgus monkeys and 10 rhesus monkeys used in this study. These results suggest that CYP1D1 plays a role as a functional, drug-metabolizing enzyme in cynomolgus monkey liver.

Keywords: Abbreviations; AHR; aryl hydrocarbon receptor; CYP; individual forms of cytochrome P450 (EC 1.14.14.1); DMSO; dimethyl sulphoxide; EROD; ethoxyresorufin; O; -deethylation; MROD; methoxyresorufin; O; -deethylation; ORF; open reading frame; P450; general term for cytochrome P450; PAH; polycyclic aromatic hydrocarbon; PCR; polymerase chain reaction; PDI; protein disulfide isomerase; RACE; rapid amplification of cDNA ends; RT; reverse transcription; SRS; substrate recognition site; XRE; xenobiotic response elementCynomolgus monkey; Cytochrome P450; Pseudogene; Genotype; Liver

Apoptosis of hepatic stellate cells mediated by specific protein nitration by Teresa Mòdol; Cristina Natal; María P. Pérez de Obanos; Eduardo Domingo de Miguel; María J. Iraburu; María J. López-Zabalza (pp. 451-458).

Inflammatory conditions are characterized by continuous overproduction of nitric oxide (NO) that can contribute to cell survival but also to cell demise by affecting apoptosis. These facts are important in regulation of hepatic fibrogenesis during exposure to inflammatory stress, since elevated NO may pose the risk of cells with a pro-fibrogenic phenotype giving rise to a sustained proliferation leading to chronic fibrosis. Since nitration of tyrosine residues occurs in a range of diseases involving inflammation, we tested the hypothesis that nitration of specific proteins could result in apoptosis of hepatic stellate cells (HSC), the primary cellular source of matrix components in liver diseases. We found the peroxynitrite generator SIN-1 to promote apoptosis in human and rat HSC, based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax protein. We also showed that SIN-1-induced apoptosis of HSC was due to protein nitration. Among the tyrosine-nitrated proteins, tyrosine kinase Lyn was identified. SIN-1 triggered a signaling pathway through Src kinase Lyn activation that resulted in increased activity of the tyrosine kinase Syk. The involvement of these signaling molecules in the apoptotic process induced by SIN-1 as well as the mechanism by which they are activated was confirmed by using specific inhibitors. In summary, NO, via protein-nitration, could play an important role in controlling liver fibrosis resolution by regulation of HSC apoptosis.

Keywords: Abbreviations; NO; nitric oxide; iNOS; inducible nitric oxide synthase; eNOS; endothelial nitric oxide synthase; cNOS; constitutive nitric oxide synthase; SIN-1; 3-morpholinosydononimide-HCl; Bax; proapoptotic member of the Bcl-2 family of proteins; NF-κB; nuclear factor kappa B; FeTMPyP; [Iron (III) tetrakis(N-methyl-4′-pyrimidyl)porphyrin-5Cl]; PP1; 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine; GSH; γ-glutamyl-cysteinyl-glycinePeroxynitrite; Programmed cell death; Inflammation; Signaling molecules; Fibrosis

Oxidative stress initiates DNA damager MNNG-induced poly(ADP-ribose)polymerase-1-dependent parthanatos cell death by Ling-Ya Chiu; Feng-Ming Ho; Shine-Gwo Shiah; Yung Chang; Wan-Wan Lin (pp. 459-470).

The alkylating agent N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG) can cause excess DNA strand breaks that lead to poly(ADP-ribose)polymerase-1 (PARP-1) overactivation and cell death (parthanatos). However, the detail mechanism of MNNG-induced parthanatos was not well-investigated. In this study, we used MNNG-treated mouse embryonic fibroblasts (MEFs) to elucidate the signaling pathways of MNNG-induced parthanatos. We found that MNNG-induced cell death accompanied by rapid PARP-1 activation, c-Jun N-terminal kinase (JNK) activation, biphasic reactive oxygen species (ROS) production and intracellular calcium increase. The early ROS production occurring at 1min and peaking at 5–15min after MNNG treatment partially resulted from NADPH oxidase. In contrast, the late phase of ROS production occurring at 30min and time-dependently increasing up to 6h after MNNG treatment was generated by mitochondria. The antioxidant, NAC can abrogate all phenomena caused by MNNG. Results indicate that the calcium rise was downstream of early ROS production, and was involved in PARP-1 and JNK activation. Moreover, the PARP inhibitor was able to reduce MNNG-induced late-phase ROS production, calcium elevation, and cell death. Results further indicated the involvement of RIP1 in sustained ROS production and calcium increase. We characterized the interactive roles of ROS, calcium, JNK, and RIP1 in MNNG-induced cell death. We found that in addition to the alkylating property previously demonstrated, ROS production triggered by MNNG results in enhanced DNA damage and PARP-1 activation. Moreover, intracellular calcium elevation and ROS production have mutual amplification effects and thus contribute to PARP-1-mediated parthanatos.

Keywords: DNA damage; ROS; PARP-1; RIP1; JNK; Parthanatos

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Biochemical Pharmacology (v.81, #3)

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Biochemical Pharmacology (v.81, #3)